Purification of kidney alkaline phosphatase

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Abstract
Kidney alkaline phosphatase was obtained in a high degree of purity and with an acceptable yield. The method of purification includes 9 steps: (1) autolysis; (2) precipitation with acetone and treatment with butanol; (3) fractionation with acetone between 0 and 45% (v/v); (4) fractionation with ammonium sulfate; (5) adsorption of impurities on MgCO3; (6) chromatography on CaPO4 columns; (7) adsorption of impurities on activated carbon; (8) fractionation with ethanol between 50 and 65% (v/v); (9) fractionation with ethanol between 54 and 62% (v/v). Chromatography on CaPO4 permits separation of several active fractions, eluted by different concentrations of phosphate ion. The existence of more than one alkaline phosphatase in the kidney seems probable. The most active product obtained liberates 166,000 g of phosphorus/minute/mg of protein N from sodium [beta]-glycerophosphate under optimum conditions. Electrophoretic analysis shows that the preparation has a purity of 85-90%. The UV-light spectrum of the purified preparation reveals the presence of a substance with an absorption maximum around 260 m[mu], which is not eliminated by dialysis at neutral or alkaline pH. Although the existence of an absorption maximum in the neighborhood of this wavelength is characteristic of pyrimidine nucleotides, it is not clear if this represents a prosthetic group or an impurity extremely difficult to eliminate.